Infections caused by multidrug resistant organisms pose special challenges to treating bacterial infections and therefore therapeutic strategies that combat bacterial virulence without aggravating drug resistance are in great demand. Gram-negative bacteria use acyl- homoserine lactone mediated quorum sensing to regulate key physiological activities that includes virulence, biofilm formation and toxin production. Bacterial AHL synthases use acyl- ACP and S-adenosyl-L- methionine to make intracellular AHL autoinducer signals. Although small molecule inhibitors for AHL synthase enzymes hold significant promise as antimicrobials in treating multidrug resistant bacterial infections, designig AHL synthase specific inhibitors does remain a significant challenge because both acyl-ACP and SAM are used as substrates by many essential eukaryotic enzymes. To ensure efficient interbacterial communication, signal-synthesizing enzymes such as AHL synthases must precisely make the native signal for that bacterium and avoid synthesizing nonspecific signals (signal fidelity). In this proposal, we will investigate how AHL synthase enzymes selectively recognize their native acyl-substrate from a pool of non-native substrates to enforce signal fidelity in bacterial quorum sensing. In particular, we will determine the extent to which each enzymatic step in AHL synthesis contributes to signal fidelity. Based on our preliminary data with Burkholderia mallei BmaI1 AHL synthase, we hypothesize that acyl-substrate recognition predominantly occurs at [Enzyme.acyl-substrate.SAM] ternary complex. We will test this hypothesis for a broad array of AHL synthase enzymes. The three aims proposed in this application should collectively provide key insights into molecular basis of acyl-ACP substrate recognition by short, medium and long-chain synthases, which will inform the design of AHL synthase specific inhibitors.